We are using a yeast system we constructed earlier for evaluating any Hsp70 isoform to investigate function of Hsp70 homologs within and across species with regard to cell growth and propagation of two different prions. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. The constitutively expressed S. cerevisiae Hsp70 isoforms Ssa1 and Ssa2 are 98% identical and the stress-inducible counterparts Ssa3 and Ssa4 share 88% identity and are 80% identical to Ssa1/2. Yarrowia lipolytica is an evolutionarily distant yeast that also contains four cytosolic Hsp70 isoforms that share 92-95% homology and are 80% identical to the S. cerevisiae Hsp70s. Although we designated them Ssa5-8 to imply similarity and to distinguish them from the S. cerevisiae Hsp70s, they are uncharacterized and it is not known which, if any, are constitutive or stress-inducible or if they overlap functionally with each other or with the S. cerevisiae Ssa proteins. Using our system we characterized several similarities and differences among these eight naturally occurring Hsp70 isoforms in supporting cell growth and propagation of different yeast prions. Our work presents the first systematic evaluation of a subfamily of essential paralogs within and between species and it provides much insight into the conservation and divergence of Hsp70 function. Our findings show that both intra- and inter-species Hsp70 isoforms have distinct activities with regard to functions in essential cellular housekeeping processes, in protecting cells from exposure to lethal heat, in refolding heat denatured protein in vivo, and in replication and growth processes necessary for propagation of yeast prions. Current work focuses on understanding the molecular bases of the differences, which could lead to design of strategies targeting Hsp70 function in vivo in ways that would hinder amyloid accumulation. We are also using our system to investigate structural bases for the functional distinctions, with an eye toward determining whether the subtle differences in structure influence intrinsic Hsp70 activities or interactions with co-chaperones that regulate Hsp70.